Progress in non-A, non-B hepatitis (NANBH) has been rapid since the successful cDNA cloning of the hepatitis C virus (HCV) genome by Houghton, et al.at Chiron. We have developed a sensitive PCR based method to detect the viral genome in clinical samples. This assay seems to be about 10 fold more sensitive than chimpanzee infectivity. We have shown that the 5' non-coding region of the HCV RNA genome is highly conserved between strains and PCR primers from this region that appear to be universal.We have developed a new diagnostic assay that combines an enzyme based detection hybridization assay with PCR in a microtiter format as a simplified method to quantitate HCV RNA in clinical samples. We have found replicate form of HCV RNA not only in the liver of infected patients and chimpanzees but also surprisingly in the serum as well. Further studies to clarify this unusual finding are in progress. We have cloned nearly the entire genome of HCV and produced a master clone of approximately 9400 nucleotides in length that includes the entire coding region. The coding region has been inserted into a vaccinia virus vector with the hopes that we can express assembled HCV particles that will be a useful immunogen.The Structural genes of HCV have been inserted into baculovirus expression systems and are being used as antigens in diagnostic assays. Studies on the protease of HCV are underway with Charles Rice in order to study the processing of the virion polyprotein and the structure and funtion of the enzyme itself. We are developing tissue culture systems in collaboration with Harry Greenberg (Stanford) and Curtis Harris (NCI). Immunopathogenesis of HCV infections is being investigated by isolating the T-cell epitopes.In collaboration with J.Berzofsky we have immunized mice with a vaccinia virus expressing the NS 5 region of HCV and test the T-cell response to synthetic peptides from the same region. To date one possible epitope has been found. We have also isolated HCV specific killer T-cell lines from liver infiltrating lymphocytes of infected chimpanzees.